Fatty acids (FAs) are activated by fatty acyl-CoA synthetase (ACS) before they enter subsequent metabolic pathways. The anaerobic protozoa Giardia, Entamoeba and Trichomonas are unable to synthesize FAs de novo. They scavenge FAs from the host and/or microenvironment, but possess ACS for FA activation and FA elongase (ELO) for limited FA extension. We discovered that the ACS inhibitor triacsin C (triaC) could inhibit the ACS enzyme activity in Giardia as well as the parasite growth in vitro, indicatin that ACSs could serve as novel drug targets. In this project, we will fully explore the potential t develop novel therapeutics against Giardia and other anaerobic protozoa by targeting parasite ACS enzymes and achieving the following aims. In the R21 phase, we will: 1) determine the biochemical features of GiACS enzymes and develop a high- throughput screening (HTS) assay to test FDA-approved drugs; 2) synthesize analogs of triaC for testing anti-GiACS efficacy and SAR analysis; 3) test the efficacy of triaC and top hits against strains from three Giardia assemblages; and 4) assess whether ACS could serve as a broad-spectrum drug target by testing the efficacy of triaC and top screening hits against other anaerobic protozoa, including Acanthamoeba, Entamoeba and Trichomonas. In the R33 phase, we will: 1) discover novel anti-GiACS inhibitors by HTS of the ActiProbe10K library; 2) synthesize more triaC analogs and analogs of top hits for testing efficacy against Giardia and other protozoa in vitro and SAR analysis, and validate the mechanism of drug action by functional genomics analysis in Giardia; 3) characterize ACSs from other protozoa that are sensitive to the inhibition of ACS for screening known drugs and SAR analysis; and 4) evaluate top hits in in vitro and in vivo pre-efficacy ADME assays and for efficacy against Giardia in an animal model. We have recently reported that ACS could serve as a novel drug target in Cryptosporidium. Our preliminary data also confirmed the efficacy of triaC on Giardia and Acanthamoeba, which strongly supports ACS as a broad-spectrum drug target in protozoan parasites. By taking advantage of our team's expertise in parasite biochemistry and medicinal chemistry, the successful completion of this project is expected with a great potential to identify a number of known drugs and novel compounds efficacious against Giardia and other anaerobic protozoa for repurposing and/or for developing new therapeutics.